Affiliation

Abstract

The hippocampus has been hypothesized to function as a "spatial" or "cognitive" map, however, the functional cellular organization of the spatial map remains a mystery. The majority of electrophysiological studies, thus far, have supported the view of a random-type organization in the hippocampus. However, using immediate early genes (IEGs) as an indicator of neuronal activity, we recently observed a cluster-type organization of hippocampal principal cells, whereby a small number ( approximately 4) of nearby cells were activated in rats exposed to a restricted part of an environment. To determine the fine structure of these clusters and to provide a 3D image of active hippocampal cells that encode for different parts of an environment, we established a functional mapping of IEGs zif268 and Homer1a, using in situ hybridization and 3D-reconstruction imaging methods. We found that, in rats exposed to the same location twice, there were significantly more double IEG-expressing cells, and the clusters of nearby cells were more "tightly" formed, in comparison to rats exposed to two different locations. We propose that spatial encoding recruits specific cell ensembles in the hippocampus and that with repeated exposure to the same place the ensembles become better organized to more accurately represent the "spatial map."

A: Environment. Behavioral testing was performed in a black painted room approximately 2.5m2 in diameter. An elevated 8-arm maze, with only two arms attached, was placed in the center of the room. The arms, which were 60-cm long, were sectioned off with a barrier in the middle of the arm and the animals were placed on Locations A or B (size: 10 × 30 cm2 each) on the end of the arms. Distal to each arm were a number of cues. The room was always illuminated with red light. During place exposure of an animal, a white light was turned on. Included in the room was the isolation chamber in which the animals were kept. B: Qualitative time course of peak expression of Homer1a and zif268 mRNAs. The differential time course of these two genes allows for identification of neuronal activity of the two exposures. C: Experimental Paradigm. Three experimental groups of animals were used – non-exposed controls (CON), exposed to two different locations once (DL), or exposed to the same location twice (SL). All animals were habituated to the arms for 15 min on two consecutive days. They were then returned to their home cage and placed in an isolation chamber for 48 hr. On the day of the experiment, they were exposed to the arms as indicated and 30 min after the initial exposure were anesthetized and sacrificed.

A-D: Autoradiographs showing expression of Homer1a (B) and zif268 (D) mRNAs with [35S]-labeled antisense and sense riboprobes for Homer1a (A) and zif268 (C). Contrary to the sense signals, strong antisense signals for Homer1a and zif268 were found in the cortex and the principal cell layers of the hippocampus. E-M: Expression of DAB-labeled Homer1a (E-G), and DAB- (H-J) and grain-labeled (K-M) zif268 in the CA1 region. There were three experimental groups: control (E,H,K), and groups exposed to one arm for 5min (5min-group, F,I,L) and exposed for 5min and kept in dark for 25min (30min-group, G,J,M). N-Q: Double-labeling ISH showing expression of DAB-labeled Homer1a (arrow) and grain-labeled zif268 (arrowheads) in CA1 pyramidal cell nuclei (violet) “on-emulsion” (N,O), and “on-tissue” (P,Q) focal planes. R-S: Expression of Homer1a (red) and zif268 (green) mRNAs in nuclei (blue) by two color-fluorescent ISH showing a similar distribution as with double DAB/grain-labeling ISH. T-U: Image of "on-emulsion" focal plane for DAB (brown) and Cresyl Violet staining. V-W: Brown DAB-labeled signals appear as white signals when the “on-emulsion” and “on-tissue” images in the same frame were subtracted (see Methods). X-Y: Signal-detection image overlaid with the on-tissue image and the subtracted image, in which the signals were colored in red. The higher magnification of the insets shows the nucleus with the staining in O, Q, S, U, W, and Y. Scale bars, A-D: 2mm; E-J: 100μm; K-M: 25μm; N,P: 20μm; R,T,V,X: 50μm.

Fig. 3. Quantification of zif268- and Homer1a-labeled…

Fig. 3. Quantification of zif268- and Homer1a-labeled CA1 neurons

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A : Line-plots showing the distribution…

Fig. 3. Quantification of zif268- and Homer1a-labeled CA1 neurons

A: Line-plots showing the distribution of the number of zif268 grains in CA1 neurons that also contain DAB-labeled Homer1a and Cresyl Violet stained nuclei in non-exposed home-cage animals (CON), exposed to two different locations once (DL), or exposed to the same location twice (SL). Arrow shows the threshold of the number of zif268 grains in the nuclei used for the double IEG-labeling analysis. B: Bar-plots showing the cell population with cumulative values of more than 5 zif268 grains in the CON, DL, and SL groups. C: Bar-plots showing the average numbers of zif268 grains in the double IEG-labeled cells between the DL and SL groups. Error bars indicate S.E.M. in each group. *p< 0.01, significant difference between control and experimental groups. †p< 0.05, significant difference between the DL and SL groups.

The upper and lower levels showing two representative 3D-reconstructed images in each group. The inset in the corner shows a lower magnification of the image. The total number of double IEG-labeled cells was 87 (A upper), 91 (A lower), 110 (B upper), and 133 (B lower) per 2.4 - 2.9 × 107 μm3 (192 μm thickness in the anterio-posterior axis by 8 sections and 1200-μm-long CA1 pyramidal cell layer in coronal section). There appears to be clusters of nearby cells in both of the DL and SL groups.

A-B: Cumulative line-plots showing the N1 distribution between real and simulated samples in the DL (A) and SL (B) groups. Histograms also show the subtracted values of the N1 between real and simulated samples (dark-gray area). C: Cumulative line-plots showing the normalized N1 (N1’) distribution between the DL and SL groups. Histograms also show the subtracted values of the N1’ between the DL and SL groups (dark-gray area). †p< 0.05, ††p< 0.005, †††p< 1 × 10-13, significant difference between pairs by Two-sample Kolmogorov-Smirnov test.

A: Representative comparisons of D1-2 (D1-2,i,j < D1-2,k,l). In animal j, cell i,j(1) is shown as the nearest neighboring cell, and Cell i,j(2) is shown as the second nearest neighboring cell from cell i, which is cell i,j(0) as a starting point. As compared to animal j, animal l has a larger gap between Cell k,l(2) and Cell k,l(0). This can be interpreted as either forming a cluster of the two nearby cells in animal l, or forming a cluster of the three nearby cells in animal j. B: Another example of comparisons of D2-3 (D2-3,i,j< D2-3,k,l). As compared to animal j, animal l has a larger gap between Cell k,l(3) and Cell k,l(0). This can be interpreted as either forming a cluster of the three nearby cells in animal l, or forming a cluster of the four nearby cells in animal j.